USPTO Patents Application 10817607

(19)

Europaisches Patentamt
European Patent Office
Office europeen des brevets

(12)

(it) EP 1 199 372 A2

EUROPEAN PATENT APPLICATION

(43) Date of publication:

24.04.2002 Bulletin 2002/17

(21) Application number: 01308837.2

(22) Date of filing: 17.10.2001

(51) Intci7: C12Q 1/68, C07K 16/28,
C07K 14/705, C12N 15/12

(84) Designated Contracting States:

(72) Inventor: Morten, John Edward Norris

AT BE CH CY DE DK ES Fl FR GB GR IE IT LI LU

Cheshire SK10 4TG (GB)

MC NL PT SE TR

Designated Extension States:

(74) Representative: Giles, Allen Frank et al

AL LT LV MK RO SI

AstraZeneca,

Global Intellectual Property Patents,

(30) Priority: 21.10.2000 GB 0025859

Mereside,

06.04.2001 GB 01 08654

Alderley Park

02.11.2000 US 244897 P

Macclesfield, Cheshire SK10 4TG (GB)

(71 ) Applicant: AstraZeneca AB

151 85 Sodertalje (SE)

(54) Polymorphisms in the human P2X7 gene

(57) This invention relates to polymorphisms in the
human P2X 7 gene and corresponding novel allelic
polypeptides encoded thereby. The invention also re-
lates to methods and materials for analysing allelic var-

iation in the P2X 7 gene, and to the use of P2X 7 polymor-
phism in treatment of diseases with P2X 7 drugs.

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EP1 199 372 A2

Description

[0001] This invention relates to polymorphisms in the human P2X 7 gene and corresponding novel allelic Polypeptides
encoded thereby. The invention also relates to methods and materials for analysing allelic variation in the P2X 7 gene,
and to the use of P2X 7 polymorphism in treatment of diseases with P2X 7 drugs.

0002] The P2X 7 receptor (previously known as P2Z receptor), which is a ligand-gated ion channel, . present on a
variety of cell types, largely those known to be involved in the inflammatory/immune process, specifically, mac ™P ha 9« s -
matf cells and lymphocytes (T and B). Activation of the P2X 7 receptor by extracellular nucleolus, in particular ade-
S,e\Jhos P hate, leads to the release of interleukin-1p (IL-1» and giant ce.l formation
ceHs), degranu.ation (mast cells) and L-se.ectin shedding (lymphocytes). P2X 7 receptors are also located o ^ antigen-
presenting cells (ARC), keratinocytes. salivary acinar cells (parotid cells) and hepatocytes. Compounds acting _at Jhe
P2X 7 receptor are therefore indicated as pharmaceuticals for use in the treatment of rheumatoid arthntis, osteoarthrjs
osor asis allergic dermatitis, asthma, chronic obstructive pulmonary disease (COPD), hyperrespons.veness of the
glomerulonephritis, irritable bowel disease. Crohn's disease, ulcerative colitis, atherosde^,
growm and metastasesof malignant cells, myoblastic leukaemia, diabetes, Alzheimer's d.sea^ meninges oWro-
sis bum injury, ischaemic heart disease, stroke and varicose veins. For further background, the reader ,s referred to
he following articles: North and Barnard in Current Opinion in Neurobiology 1 997. 7 ,346^357; Rassendren, JBC 1 9 £
273, 5482-6; and Buell, Receptors and Channels, 1998, 5, 347-354. The terms P2X 7 , P2X 7 receptor and P2RX7 are

"SSSTSSSSiZS:^ polymorphisms in the 5" UTR region of the P2X 7 polynucleotide relate to the position
in SEQ ID NO 1 unless stated otherwise or apparent from the context.

[0004] All positions herein of polymorphisms in the exon regions of the P2X 7 polynucleotide relate to the position in
SEQ ID NO 2 unless stated otherwise or apparent from the context. nnB - Mnn
[0005] All positions herein of polymorphisms in the intron regions of the P2X 7 P olynucleot,de relate to the position
in SEQ ID NO 3 unless stated otherwise or apparent from the context. . Bminun ., ,„ looe

[0006] All positions herein of polymorphisms in the P2X 7 polypeptide relate to the pos,t,on ,n SEQ ID NO 4 unless
stated otherwise or apparent from the context.

[0007] One approach is to use knowledge of polymorphisms to help identify patients most suited to therapy with
particular pharmaceutical agents (this is often termed "pharmacogenetics"). Pharmacogenetics can also be used in
pharmaceutical research to assist the drug selection process. Polymorphisms are used in mapping the human genome
and to elucidate the genetic component of diseases. The reader is directed to the following references for background
delate on pharmacogenetics and other uses of polymorphism detection: Under eta,. (1997), Cl.n.cal Chemistry, 43,
254 Marshall 0997) Nature Biotechnology, 15, 1249; .nternational Patent Application WO 97/40462, Spectra Bio-
medical- and Schafer ef al. (1 998), Nature Biotechnology, 16, 33.

m008] Clinical trials have shown that patient response to treatment with pharmaceuticals is often heterogeneous.
Thus there is a need for improved approaches to pharmaceutical agent design and therapy.
[0009] Point mutations in polypeptides will be referred to as follows: natural amino ac.d (us.ng 1 or 3 letter
Zenclatu e) , position, new amino acid. For (a hypothetical) example "D25K" or "Asp25Lys" means that at position
25 an aspartic acid (D) has been changed to lysine (K). Multiple mutations in one polypept.de will be shown between
square brackets with individual mutations separated by commas. „ H , hirh ,
[0010] The present invention is based on the discovery of polymorphisms in P2X 7 . In part.cular, we have found thirty
polymorphisms in the coding sequence of the P2X 7 gene, 1 2 of which lead to changes in the sequence of expressed

According to one aspect of the present invention there is provided a method for the diagnosis of a polymor-
phism in P2X 7 in a human, which method comprises determining the sequence of the human at at least one polymorphic
poslon and determining the status of the human by reference to polymorphism in P2X 7 . Preferred polymorph* pos-
tions are one or more of the following positions:

positions 936, 1 01 2, 1 1 47, 1 343 and 1 476 in the 5'UTR region of the P2X 7 gene as defined by the position in SEQ

p D osTons253 488, 489, 760, 835. 853, 1068, 1096, 1315, 1324, 1405, 1448, 1494, 1513, 1628 and 1772 in the
coding region of the P2X 7 gene as defined by the position in SEQ ID NO: 2; and

positions 4780, 4845, 4849, 5021, 5554, 5579, 5535, 5845 and 6911 in the mtron region of the P2X 7 gene as
defined by the position in SEQ ID NO: 3;

positions 76 ,155, 245, 270, 276, 348, 357, 430, 433, 460, 490 and 496 in the P2X7 polypeptide as defined by the
position in SEQ ID NO: 4.

[0012] The term human includes both a human having or suspected of having a P2X 7 mediated disease and an

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EP 1 199 372 A2

asymptomatic human who may be tested for predisposition or susceptibility to such disease. At each position the
human may be homozygous for an allele or the human may be a heterozygote.

[0013] The term "status" refers to the genetic status of the human as detected by potential sequence variation at
defined positions of a polynucleotide or corresponding protein. The term "diagnosis of a polymorphism" refers to de-
5 termination of the genetic status of an individual at a polymorphic position (in which the indiviual may be homozygous
or heterozygous at each position).

[0014] The term polymorphism includes single nucleotide substitution, nucleotide insertion and nucleotide deletion
which in the case of insertion and deletion includes insertion or deletion of one or more nucleotides at a position of a
gene and corresponding alterations in expressed protein.
w [001 5] In one embodiment of the invention preferably the method for diagnosis described herein is one in which the
polymorphism in the in the 5'UTR region of the P2X 7 gene as defined by the position in SEQ ID NO: 1 is any one of
the following:

at position 936 is presence of C and/or A; at position 1012 is presence of T and/or C;
15 at position 1 1 47 is presence of A and/or G; at position 1 343 is presence of G and/or A; and

at position 1 476 is presence of A and/or G.

[0016] In one embodiment of the invention preferably the method for diagnosis described herein is one in which the
polymorphism in the coding region of the P2X 7 gene as defined by the position in SEQ ID NO: 2 is any one of the
20 following:

at position 253 is presence of T and/or C; at position 488 is presence of G and/or A;
at position 489 is presence of C and/or T; at position 760 is presence of T and/or G;
at position 835 is presence of G and/or A; at position 853 is presence of G and/or A;
25 at position 1 068 is presence of G and/or A; at position 1 096 is presence of C and/or G;

at position 1315 is presence of C and/or G; at position 1324 is presence of C and/or T;
at position 1405 is presence of A and/or G; at position 1448 is presence of C and/or T;
at position 1494 is presence of A and/or G; at position 1513 is presence of A and/or C;
at position 1628 is presence of G and/or T; and at position 1772 is presence of G and/or A.

30

[0017] In one embodiment of the invention preferably the method for diagnosis described herein is one in which the
polymorphism in the intron region of the P2X 7 gene as defined by the position in SEQ ID NO: 3. is any one of the
following:

35 at position 4780 is presence of C and/or T; at position 4845 is presence of C and/or T;

at position 4849 is presence of A and/or C; at position 5021 is presence of T and/or C;

at position 5554 is presence of 3 and/or 4 repeats of GTTT (wherein position 5554 refers to the position of the G
in the first unit repeat);

at position 5579 is presence of G and/or C; at position 5535 is presence of A and/or T;
^o at position 5845 is presence of C and/or T; and at position 6911 is presence of T and/or C.

[0018] In one embodiment of the invention preferably the method for diagnosis described herein is one in which the
polymorphism in the P2X 7 protein as defined by the position in SEQ ID NO: 4. is any one of the following: val76ala,
his155tyr, val245gly, arg270his, arg276his, ala348thr, thr357ser, pro430arg, ala433val, gln460arg, ser490gly and
45 glu496ala.

[0019] The method for diagnosis is preferably one in which the sequence is determined by a method selected from
amplification refractory mutation system, restriction fragment length polymorphism and primer extension.
[0020] The status of the individual may be determined by reference to allelic variation at any 1 , 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more positions.
so [0021] The test sample of nucleic acid is conveniently a sample of blood, bronchoalveolar lavage fluid, sputum, or
other body fluid or tissue obtained from an individual. It will be appreciated that the test sample may equally be a nucleic
acid sequence corresponding to the sequence in the test sample, that is to say that all or a part of the region in the
sample nucleic acid may firstly be amplified using any convenient technique e.g. PCR, before analysis of allelic vari-
ation.

55 [0022] It will be apparent to the person skilled in the art that there are a large number of analytical procedures which
may be used to detect the presence or absence of variant nucleotides at one or more polymorphic positions of the
invention. In general, the detection of allelic variation requires a mutation discrimination technique, optionally an am-
plification reaction and optionally a signal generation system. Table 1 lists a number of mutation detection techniques,

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EP1 199 372 A2

some based on the PCR These may be used in combination with a number of signal generation systems, a selection

"Laboratory Protocols for Mutation Detection", Ed. by U. Landegren, Oxford Un.vers.ty Press, 1996 and PCR , 2
Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Abbreviations:

[0023]

10

15

20

25

30

35

40

ALEX™

Amplification refractory mutation system linear extension

APEX

Arrayed primer extension

ARMS™

Amplification refractory mutation system

b-DNA

Branched DNA

bp

base pair

CMC

Chemical mismatch cleavage

COPS

Competitive oligonucleotide priming system

DGGE

Denaturing gradient gel electrophoresis

ELISA

Enzyme Linked Immuno Sorbent Assay

FRET

Fluorescence resonance energy transfer

LCR

Ligase chain reaction

MASDA

Multiple allele specific diagnostic assay

NASBA

Nucleic acid sequence based amplification

OLA

Oligonucleotide ligation assay

PCR

Polymerase chain reaction

PTT

Protein truncation test

RFLP

Restriction fragment length polymorphism

SDA

Strand displacement amplification

SNP

Single nucleotide polymorphism

SSCP

Single-strand conformation polymorphism analysis

SSR

Self sustained replication

TGGE

Temperature gradient gel electrophoresis

45

50

55

Table 1 -

Mutation Detection Techniques

General: DNA sequencing, Sequencing by hybridisation

Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase, Heteroduplex analysis, CMC, Enzymatic mismatch cleavage

Hybr sSS phas^Sridisation: Dot blots, MASDA, Reverse dot blots, Oligonucleotide arrays (DNA Chips).

Solution phase hybridisation: Taqman™ - US-5210015 & US-5487972 (Hoffmann-La Roche , Molecular
Beacons - Tyagi ef a/ (1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public Health Inst., New York)
Extension Based: ARMS™, ALEX™ - European Patent No. EP 332435 B1 (Zeneca Limited), COPS - Gibbs etal
(1989), Nucleic Acids Research, 17, 2347.
Incorporation Based: Mini-sequencing, APEX
Restriction Enzyme Based: RFLP, Restriction site generating PCR
* Note: not useful for detection of promoter polymorphisms.

4

EP1 199 372 A2

Table 1 - (continued)

Mutation Detection Techniques
Ligation Based: OLA
Other: Invader assay

Table 2 -

Signal Generation or Detection Systems
w Fluorescence: FRET, Fluorescence quenching, Fluorescence polarisation - United Kingdom Patent No. 2228998
(Zeneca Limited)

Other: Chemiluminescence, Electrochemiluminescence, Raman, Radioactivity, Colorimetric, Hybridisation
protection assay, Mass spectrometry

15

20

25

Table 3 -
Further Amplification Methods
SSR, NASBA, LCR, SDA, b-DNA

Table 4-

Protein variation detection methods
Immunoassay
Immunohistology
Peptide sequencing

[0024] Preferred mutation detection techniques include ARMS™, ALEX™, COPS, Taqman, Molecular Beacons,
RFLP, and restriction site based PCR and FRET techniques. Immunoassay techniques are known in the art e.g. A
Practical Guide to ELISA by D M Kemeny, Pergamon Press 1 991 ; Principles and Practice of Immunoassay, 2 nd edition,
C P Price & D J Newman, 1997, published by Stockton Press in USA & Canada and by Macmillan Reference in the
United Kingdom. Histological techniques are described in Theory and Practice of Histological Techniques by J D Ban-
croft & A Stevens, 4 th Edition, Churchill Livingstone, 1996. Protein sequencing is described in Laboratory Techniques
in Biochemistry and Molecular Biology, Volume 9, Sequencing of Proteins and Peptides, G Allen, 2 nd revised edition,
Elsevier, 1989. Particularly preferred methods include ARMS™ and RFLP based methods. ARMS™ is an especially
preferred method.

[0025] In a further aspect, the diagnostic methods of the invention are used to assess the pharmacogenetics of a
drug acting at P2X 7 .

[0026] Assays, for example reporter-based assays, may be devised to detect whether one or more of the above
polymorphisms affect transcription levels and/or message stability.

[0027] Individuals who carry particular allelic variants of the P2X 7 gene may therefore exhibit differences in their
ability to regulate protein biosynthesis under different physiological conditions and will display altered abilities to react
to different diseases. In addition, differences arising as a result of allelic variation may have a direct effect on the
response of an individual to drug therapy. The diagnostic methods of the invention may be useful both to predict the
clinical response to such agents and to determine therapeutic dose.

[0028] In a further aspect, the diagnostic methods of the invention, are used to assess the predisposition and/or
susceptibility of an individual to diseases mediated by P2X 7 . This may be particularly relevant in the development of
hyperlipoproteinemia and cardiovascular disease and the present invention may be used to recognise individuals who
are particularly at risk from developing these conditions.

[0029] In a further aspect, the diagnostic methods of the invention are used in the development of new drug therapies
which selectively target one or more allelic variants of the P2X 7 gene. Identification of a link between a particular allelic
variant and predisposition to disease development or response to drug therapy may have a significant impact on the
design of new drugs. Drugs may be designed to regulate the biological activity of variants implicated in the disease
process whilst minimising effects on other variants.

[0030] In a further diagnostic aspect of the invention the presence or absence of variant nucleotides is detected by
reference to the loss or gain of, optionally engineered, sites recognised by restriction enzymes.
[0031] According to another aspect of the present invention there is provided a human P2X 7 gene or its complemen-
tary strand comprising a variant allelic polymorphism at one or more of positions defined herein or a fragment thereof

5

EP1 199 372 A2

of at least 20 bases comprising at least one novel polymorphism.

[0032] Fragments are at least 17 bases, more preferably at least 20 bases, more preferably at least 30 bas
[0033] According to another aspect of the present invention there is provided a polynucleotide comprising
20 bases of the human P2X 7 gene and comprising a polymorphism selected from any one of the following:

Region

Polymorphism SEQ ID NO: 1

S'UTR

936 C->A

1012 T^C

1147 A->G

1343 G->A

1476 A->G

Region

Polymorphism SEQ ID NO: 2

exon 2

253 T->C

exon 5

488 G-*A

489 C->T

exon 7

760 T->G

exon 8

835 G->A

853 G->A

exon 11

1068 G->A

1096 C->G

exon 12

1315C->G

exon 13

1324 C->T

1405 A^G

1448 C->T

1494 A->G

1513 A^C

1628 G->T

1772 G->A

Region

Polymorphism SEQ ID NO: 3

intron E

4780 C->T

4845 C-+T

4849 A->C

intron F

5021 T-»C

5554 (GTTT)n=3,4

5579 G->C

5535 A->T

intron G

5845 C-»T

6911 T->C

[0034] According to another aspect of the present invention there is provided a polynucleotide comprising
20 bases of the human P2X 7 gene and comprising an allelic variant selected from any one of the following:

Region

Variant SEQ ID NO: 1

5'UTR

936 A
1012C

6

EP1 199 372 A2

(continued)

5

Region

Variant SEQ ID NO: 1

1147 G
1343 A
1476G

10

15

20

25

30

Region

Variant SEQ ID NO: 2

exon 2

253 C

exon 5

488 A

489 T

exon 7

760 G

exon 8

835 A

853 A

exon 11

1068 A

1096 G

exon 12

1315G

exon 13

1324T

1405G

1448T

1494 G

1513C

1628 T

1772 A

35

40

Region

Variant SEQ ID NO: 3

intron E

4780 T

4845 T

4849 C

intron F

5021 C

5554 (GTTT) n ,n=4

5579 C

5535 T

intron G

5845 T

6911 C

[0035] According to another aspect of the present invention there is provided a human P2X 7 gene or its complemen-
tary strand comprising a polymorphism, preferably corresponding with one or more the positions defined herein or a
fragment thereof of at least 20 bases comprising at least one polymorphism.

[0036] Fragments are at least 17 bases, more preferably at least 20 bases, more preferably at least 30 bases.
[0037] The invention further provides a nucleotide primer which can detect a polymorphism of the invention.
[0038] According to another aspect of the present invention there is provided an allele specific primer capable of
detecting a P2X 7 gene polymorphism, preferably at one or more of the positions as defined herein.
[0039] An allele specific primer is used, generally together with a constant primer, in an amplification reaction such
as a PCR reaction, which provides the discrimination between alleles through selective amplification of one allele at
a particular sequence position e.g. as used for ARMS™ assays. The allele specific primer is preferably 17- 50 nucle-
otides, more preferably about 17-35 nucleotides, more preferably about 17-30 nucleotides.

[0040] An allele specific primer preferably corresponds exactly with the allele to be detected but derivatives thereof

7

EP1 199 372 A2

are also contemplated wherein about 6-8 of the nucleotides at the 3' terminus correspond with the allele to be detected
and wherein up to 10, such as up to 8, 6, 4, 2, or 1 of the remaining nucleotides may be varied without significantly
affecting the properties of the primer.

[0041] Primers may be manufactured using any convenient method of synthesis. Examples of such methods may
5 be found in standard textbooks, for example "Protocols for Oligonucleotides and Analogues; Synthesis and Properties,"
Methods in Molecular Biology Series; Volume 20; Ed. Sudhir Agrawal, Humana ISBN: 0-89603-247-7; 1 993; 1 st Edition.
If required the primer(s) may be labelled to facilitate detection.

[0042] According to another aspect of the present invention there is provided an allele-specific oligonucleotide probe
capable of detecting a P2X 7 gene polymorphism, preferably at one or more of the positions defined herein.
10 [0043] The allele-specific oligonucleotide probe is preferably 17- 50 nucleotides, more preferably about 17-35 nu-
cleotides, more preferably about 17-30 nucleotides.

[0044] The design of such probes will be apparent to the molecular biologist of ordinary skill. Such probes are of any
convenient length such as up to 50 bases, up to 40 bases, more conveniently up to 30 bases in length, such as for
example 8-25 or 8-15 bases in length. In general such probes will comprise base sequences entirely complementary
is to the corresponding wild type or variant locus in the gene. However, if required one or more mismatches may be
introduced, provided that the discriminatory power of the oligonucleotide probe is not unduly affected. The probes of
the invention may carry one or more labels to facilitate detection.

[0045] According to another aspect of the present invention there is provided an allele specific primer or an allele
specific oligonucleotide probe capable of detecting a P2X 7 gene polymorphism at one of the positions defined herein.
20 [0046] According to another aspect of the present invention there is provided a diagnostic kit comprising an allele
specific oligonucleotide probe of the invention and/or an allele-specific primer of the invention.
[0047] The diagnostic kits may comprise appropriate packaging and instructions for use in the methods of the inven-
tion. Such kits may further comprise appropriate buffer(s) and polymerase(s) such as thermostable polymerases, for
example taq polymerase.

25 [0048] In another aspect of the invention, the polymorphisms of this invention may be used as genetic markers in
linkage studies. This particularly applies to the polymorphisms of relatively high frequency. The P2X 7 gene is on chro-
mosome 12q24 (Buell et al, Receptors and Channels, 1998, 5,347-354). Low frequency polymorphisms may be par-
ticularly useful for haplotyping as described below. A haplotype is a set of alleles found at linked polymorphic sites
(such as within a gene) on a single (paternal or maternal) chromosome. If recombination within the gene is random,

30 there may be as many as 2 n haplotypes, where 2 is the number of alleles at each SNP and n is the number of SNPs.
One approach to identifying mutations or polymorphisms which are correlated with clinical response is to carry out an
association study using all the haplotypes that can be identified in the population of interest. The frequency of each
haplotype is limited by the frequency of its rarest allele, so that SNPs with low frequency alleles are particularly useful
as markers of low frequency haplotypes. As particular mutations or polymorphisms associated with certain clinical

35 features, such as adverse or abnormal events, are likely to be of low frequency within the population, low frequency
SNPs may be particularly useful in identifying these mutations (for examples see: Linkage disequilibrium at the cys-
tathionine beta synthase (CBS) locus and the association between genetic variation at the CBS locus and plasma
levels of homocysteine. Ann Hum Genet (1998) 62:481-90, De Stefano V, Dekou V, Nicaud V, Chasse JF, London J,
Stansbie D, Humphries SE, and Gudnason V; and Variation at the von willebrand factor (vWF) gene locus is associated

40 with plasma vWF:Ag levels: identification of three novel single nucleotide polymorphisms in the vWF gene promoter.
Blood (1999) 93:4277-83, Keightley AM, Lam YM, Brady JN, Cameron CL, Lillicrap D).

[0049] According to another aspect of the present invention there is provided a computer readable medium compris-
ing at least one novel sequence of the invention stored on the medium. The computer readable medium may be used,
for example, in homology searching, mapping, haplotyping, genotyping or pharmacogenetic analysis.
45 [0050] According to another aspect of the present invention there is provided a method of treating a human in need
of treatment with a drug acting at P2X 7 in which the method comprises:

i) diagnosis of a polymorphism in P2X 7 in the human, which diagnosis preferably comprises determining the sequence
at one or more of the following positions:

50

[0051]

positions 936, 1012, 1147, 1343 and 1476 in the 5'UTR region of the P2X 7 gene as defined by the position in SEQ
ID NO: 1;

55 positions 253, 488, 489, 760, 835, 853, 1068, 1096, 1315, 1324, 1405, 1448, 1494, 1513, 1628 and 1772 in the

coding region of the P2X 7 gene as defined by the position in SEQ ID NO: 2; and

positions 4780, 4845, 4849, 5021, 5554, 5579, 5535, 5845 and 6911 in the intron region of the P2X 7 gene as
defined by the position in SEQ ID NO: 3; and

8

EP1 199 372 A2

positions 76 ,1 55, 245, 270, 276, 348, 357, 430, 433, 460, 490 and 496 in the P2X 7 polypeptide as defined by the
position in SEQ ID NO: 4;

and determining the status of the human by reference to polymorphism in P2X 7 ; and

5 il) administering an effective amount of the drug.

[0052] Preferably determination of the status of the human is clinically useful. Examples of clinical usefulness include
deciding which drug or drugs to administer and/or in deciding on the effective amount of the drug or drugs. The term
"drug acting at P2X7" means that drug binding with P2X7 in humans is an important part of a drug exerting its pharm-

10 ceutical effect in man. Compounds which are known to be antagonists of the P2X7 receptor are described in published
PCT application nos. WO 99/29660, WO 99/29661, WO 99/29686, WO 00/61569, WO 00/71529, WO 01/42194, WO
01/44170, WO 01/44213 and WO 01/46200. According to another aspect of the present invention there is provided
use of a drug acting at P2X 7 in preparation of a medicament for treating a disease in a human diagnosed as having a
polymorphism therein, preferably at one or more of the positions defined herein.

15 [0053] According to another aspect of the present invention there is provided a pharmaceutical pack comprising
P2X 7 drug and instructions for administration of the drug to humans diagnostically tested for a polymorphism therein,
preferably at one or more of the positions defined herein.

[0054] According to another aspect of the present invention there is provided an allelic variant of human P2X 7
polypeptide comprising at least one of the following:

20

a alanine at position 76 of SEQ ID NO 4;

a tyrosine at position 1 55 of SEQ ID NO 4;

a glycine at position 245 of SEQ ID NO 4;

a histidine at position 270 of SEQ ID NO 4;
25 a histidine at position 276 of SEQ ID NO 4;

a threonine at position 348 of SEQ ID NO 4;

a serine at position 357 of SEQ ID NO 4;

a arginine at position 430 of SEQ ID NO 4;

a valine at position 433 of SEQ ID NO 4;
30 a arginine at position 460 of SEQ ID NO 4;

a glycine at position 490 of SEQ ID NO 4; and

a glutamic acid at position 496 of SEQ ID NO 4;

or a fragment thereof comprising at least 10 amino acids provided that the fragment comprises at least one allelic
35 variant.

[0055] Fragments of polypeptide are at least 1 0 amino acids, more preferably at least 1 5 amino acids, more preferably
at least 20 amino acids.

[0056] According to another aspect of the present invention there is provided an antibody specific for an allelic variant
of human P2X 7 polypeptide as described herein.

40 [0057] Antibodies can be prepared using any suitable method. For example, purified polypeptide may be utilized to
prepare specific antibodies. The term "antibodies" is meant to include polyclonal antibodies, monoclonal antibodies,
and the various types of antibody constructs such as for example F(ab') 2 , Fab and single chain Fv. Antibodies are
defined to be specifically binding if they bind the allelic variant of P2X 7 with a K a of greater than or equal to about 1 0 7
M -1 . Affinity of binding can be determined using conventional techniques, for example those described by Scatchard

45 et al_, Ann. N. Y. Acad. Sci, §1:660 (1 949).

[0058] Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats,
sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art. In general, antigen is
administered to the host animal typically through parenteral injection. The immunogenicity of antigen may be enhanced
through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immuniza-

50 tions, small samples of serum are collected and tested for reactivity to antigen. Examples of various assays useful for
such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring
Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radi-
oimmunoassay, radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sand-
wich assays, see U.S. Patent Nos. 4,376,110 and 4,486,530.

55 [0059] Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the proce-
dures described in U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybri-
domas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980).
[0060] The monoclonal antibodies of the invention can be produced using alternative techniques, such as those

9

EP1 199 372 A2

described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas , \ Strat-
egies in Molecular Biology 3: 1-9 (1990) which is incorporated herein by reference. Similarly, binding partners can be
constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific
binding antibody. Such a technique is described in Larrick et aL, Biotechnology, 7: 394 (1989).
[0061] Once isolated and purified, the antibodies may be used to detect the presence of antigen in a sample using
established assay protocols, see for example "A Practical Guide to EUSA" by D. M. Kemeny, Pergamon Press, Oxford,
England.

[0062] According to another aspect of the invention there is provided a diagnostic kit comprising an antibody of the
invention.

[0063] According to another aspect of the present invention there is provided a polynucleotide comprising any one
of the following twenty six P2X 7 haplotypes:

1012

489

5579

835

853

1068

1096

1405

1513

SEQID
1

SEQID
2

SEQID

3

SEQID
2

SEQ ID 2

SEQ ID 2

SEQ ID 2

SEQ ID 2

SEQ ID 2

1

T

T

C

G

G

A

G

A

A

2

C

C

G

G

G

G

C

A

A

3

C

C

C

A

G

G

C

A

A

O

4

c

T

G

G

G

A

C

G

A

A

5

c

C

G

G

G

A

G

A

A

A

6

c

c

C

A

G

G

C

A

A

A

7

T

T

G

G

G

A

C

G

A

A

8

c

T

C

G

G

G

C

A

A

A

9

c

C

C

G

G

A

C

A

A

10

c

T

G

G

G

G

C

A

11

T

C

G

G

G

A

C

A

A

A

12

c

T

C

G

G

G

C

A

c

13

T

C

C

G

G

A

C

A

A

A

14

T

r±

n.

G

c

A

c

15

C

T

C

G

G

A

C

A

A

16

T

T

C

G

G

A

C

G

A

17

C

C

G

G

G

A

C

G

A

18

T

C

G

A

A

G

c

A

A

19

C

c

C

G

G

G

G

A

A

20

T

c

C

G

G

G

G

A

A

21

C

T

C

A

G

G

C

A

A

22

C

c

C

G

G

G

C

A

C

23

c

T

G

G

A

A

G

G

A

24

T

T

G

G

G

A

G

G

A

25

c

T

C

G

G

G

G

A

A

26

c

C

C

G

G

G

C

A

A

[0064] According to another aspect of the present invention there is provided a human P2X 7 polypeptide comprising
one of the following eighteen combinations of alieleic variant determined amino acids based on positions identified in
SEQ ID NO: 4:

10

EP1 199 372 A2

10

20

25

155

270

276

348

357

460

496

1

Y

R

R

T

S

Q

E

2

Y

R

R

T

T

R

E

3

Y

R

R

T

T

Q

E

4

Y

R

R

T

S

R

E

5

Y

R

R

A

T

Q

A

6

Y

R

R

A

T

Q

E

7

Y

R

R

A

S

Q .

E

8

Y

R

H

T

S

R

E

9

Y

H

R

A

T

Q

E

10

H

R

R

T

T

Q

E

11

H

R

R

T

T

R

E

12

H

R

R

A

T

Q

A

13

H

R

R

A

S

Q

E (

14

H

R

R

A

T

Q

E

15

H

R

R

T

S

Q

E

16

H

H

R

A

T

Q

A

17

H

H

R

A

T

Q

E

18

H

H

H

A

T

Q

E

[0065] According to another aspect of the present invention there is provided a polynucleotide which encodes any
human P2X 7 polypeptide combination of allelic variants defined herein.

[0066] The invention will now be illustrated but not limited by reference to the following Examples. All temperatures
are in degrees Celsius.

[0067] In the Examples below, unless otherwise stated, the following methodology and materials have been applied.
35 [0068] AMPLITAQ™ .available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase.
[0069] General molecular biology procedures can be followed from any of the methods described in "Molecular Clon-
ing - A Laboratory Manual" Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) or
"Current Protocols in Molecular Biology", Volumes 1-3 , Edited by F M Asubel, R Brent & R E Kingston, published by
John Wiley, 1998.

40 [0070] Electropherograms were obtained in a standard manner: data was collected by ABI377 data collection soft-
ware and the wave form generated by ABI Prism sequencing analysis (2.1.2).

Example 1

45 Identification of Polymorphisms

1. Methods

DNA Preparation

50

[0071] DNA was prepared from frozen blood samples collected in EDTA following protocol I (Molecular Cloning: A
Laboratory Manual, p392, Sambrook, Fritsch and Maniatis, 2 nd Edition, Cold Spring Harbor Press, 1989) with the
following modifications. The thawed blood was diluted in an equal volume of standard saline citrate instead of phosphate
buffered saline to remove lysed red blood cells. Samples were extracted with phenol, then phenol/chloroform and then
55 chloroform rather than with three phenol extractions. The DNA was dissolved in deionised water.

11

EP1 199 372 A2

Template Preparation

[0072] Templates were prepared by PCR using the oligonucleotide primers and annealing temperatures set out be-
low The extension temperature was 72° and denaturation temperature 94°. Generally 50 ng of genomic DNA was
5 used in each reaction and subjected to 35 cycles of PCR. Where described below, the primary fragment was diluted
1/100 and two microlitres were used as template for amplification of secondary fragments. PCR was performed in two
stages (primary fragment then secondary fragment) to ensure specific amplification of the desired target sequence.

Polymorphisms in P2X 7

10

[0073]

Region

Size

Polymorphism

protein change

frequency

15
20

5'UTR

936 C->A
1012T->C
1147 A->G

1343 G->A

14/ D M —

3/56
42/56
3/56
2/52
35/52

exon 1

146bp

intron A

21.7kb

exon 2

168bp

253 T->C

va!76ala

2/54

25

intron B

1.1 kb

axon 3

68bp

intron C

4.7kb

30

exon 4

/ oup

intron D

1.5kb

axon 5

95bp

488 G->A

489 C-»T

silent
his1 55tyr

17/38

35

intron E

4780 C->T
4845 C->T
4849 A-»C

39/52
39/52
28/36

exon 6

80bp

40

intron F

617bp

5021 T->C
5554 (GTTT)n=3,4
5579 G->C
5535 A->T

1/34
n=3, 14/40
26/40
1/44

45

exon 7

129bp

760 T->G

val245gly

1/40

intron G

1.3kb

5845 C->T
6911 T->C

2/40
33/50

50

exon 8

136bp

835 G->A
853 G->A

arg270hls
arg276his

16/52
1/54

intron H

exon 9

91 bp

55

intron I

1.7kb

exon 10

64bp

intron J

84bp

12

EP1 199 372 A2

(continued)

Region

Size

Polymorphism

protein change

frequency

exon 11

149bp

1068 G->A

ala348thr

18/62

1 096 C-»G

thr357ser

5/66

intron K

exon 12

101 bp

1315 C-»G

pro430arg, splice site

4/66

intron L

3.8kb

exon 1 3

497bp

1324 C->T

ala433val

1/54

1405 A-M3

gln460arg

3/54

1448 C-*T

silent

2/54

1494 A-»G

ser490gly

2/54

1513 A^C

glu496ala

8/54

1628 G->T

silent

2/52

1772 G->A

silent

24/54

Positions in the 5' UTR refer to SEQ ID NO: 1 .
Positions in exons refer to SEQ ID NO: 2.
Positions in introns refer to SEQ ID NO: 3.
Positions in protein refer to SEQ ID NO: 4.

[0074] Evidence for effects of some polymorphisms on transcription are as follows. C at position 1012 SEQ ID No 1

disrupts the TCAAT motif from an enhancer binding sequence reported in intron 1 of EGFR. A at position 1147 SEQID

No 1 disrupts the reverse sequence of the TCCTGC motif which is also an enhancer binding sequence from intron 1

EGFR. (Maekawa T, Imamoto R, Merlino G. T., Pastan I., Ishii S.

Cooperative Function of Two Separate Enhancers of RT the Human Epidermal Growth

Factor Receptor Proto-oncogene J. BioL Chem. 264:5488-5494 (1989)).

Example 2

Haplotype analysis

a) The following allele frequencies were determined in a Swedish population.
[0075]

SEQ ID NO

Position

Frequency

1

1012

46/60

2

489

27/60

3

5579

39/60

2

835

16/58

2

853

3/60

2

1068

24/58

2

1096

6/58

2

1045

11/60

2

1513

10/60

b) Haplotype data.

[0076] Analysis of 1 5 Swedish families with at least one asthmatic child using primer extension (SNapShot™, Perkin

13

EP 1 199 372 A2

Elmer) genotyping and GeneHunter™ analysis demonstrated the following haplotypes:

1012

489

5579

835

853

1068

1096

1405

1513

Frequency n/58

1

T

T

C

G

G

A

G

A

A

i

2

C

C

G

G

G

G

C

A

A

3

3

C

C

C

A

G

G

C

A

C

■4
1

4

c

T

G

G

G

A

C

G

A

5

5

c

C

G

G

G

A

G

A

A

1

6

c

C

C

A

G

G

C

A

A

8

7

T

T

G

G

G

A

C

G

A

1

8

c

T

C

G

G

G

C

A

A

3

9

c

C

C

G

G

A

C

A

A

3

10

c

T

G

G

G

G

C

A

C

2

11

T

C

G

G

G

A

C

A

A

2

12

c

T

C

G

G

G

C

A

C

3

13

T

C

C

G

G

A

C

A

A

4

14

T

C

C

G

G

G

c

A

C

1

15

C

T

C

G

G

A

c

A

A

2

16

T

T

C

G

G

A

c

G

A

1

17

C

C

G

G

G

A

c

G

A

2

18

T

C

G

A

A

G

c

A

A

2

19

C

c

C

G

G

G

G

A

A

1

20

T

c

C

G

G

G

G

A

A

1

21

C

T

C

A

A

va

c

A

A

4

22

c

C

c

G

G

G

C

A

C

3

P3

c

T

G

G

A

A

G

G

A

1

24

T

T

G

G

G

A

G

G

A

1

25

c

T

C

G

G

G

G

A

A

1

26

c

C

c

G

G

G

C

A

A

1

This results in the following proteins:

position SEQ ID NO 4

155

270

276

348

357

460

496

Frequency N/58

amino acid

Y

R

R |

T

S

Q

E

1

Y

R

R

T

T

R

E

7

Y

R

R

T

T

Q

E

2

Y

R

R

T

S

R

E

1

Y

R

R

A

T

Q

A

5

Y

R

R

A

T

Q

E

3

Y

R

R

A

S

Q

E

1

Y

R

H

T

S

R

E

1

14

EP1 199 372 A2

(continued)

position SEQ ID NO 4

155

270

276

348

357

460

496

Frequency N/58

Y

H

R

A

T

Q

E

4

H

R

R

T

T

Q

E

9

H

R

R

T

T

R

E

2

H

R

R

A

T

Q

A

4

H

R

R

A

S

Q

E

3

H

R

R

A

T

Q

E

3

H

R

R

T

S

Q

E

1

H

H

R

A

T

Q

A

1

H

H

R

A

T

Q

E

8

H

H

H

A

T

Q

E

2

c) Analysis

[0077] Ben J. Gu, Weiyi Zhang, Rebecca A. Worthington, Ronald Sluyter, Phuong Dao-Ung, Steven Petrou, Julian
A. Barden, and James S. Wiley, J. BioL Chem. (2001) 276: 11135-11142 reported that Ala at 496 (C at 1513) leads to
loss of function in P2X7. Only one polymorphism was reported since they only analysed the final exon for SNPs

15

EP1 199 372 A2

SEQUENCE LISTING

<110> AstraZeneca AB
<120> Chemical Compounds

<130> morten

<140>
<U1>

<160> 4

<170> Patentln Ver. 2.1

<210> 1

<211> 4900

<212> DNA

<213> Homo sapiens

™; = = =; := := s.

ZZZL »««»«™ ■»""•"* ■•""*"" Z

^nt-rrrcta tatgcaactg agaagggcag ggccagggag tcacgtcca

™: — : •« — ™, -

™ — ~: r= ~ = "°
r;x irrr.; :r.rr. ~i „< «

= ; = ;= := == =
= = =: = = r
i= := = = =: i™ -
== =: s= = == =
i= =; — =i = =l- =

! otLtataca tcaggggctg aataaagggt tgtagaaatg aatgaatcaa 1320
ttacccagag ctcctataca t« atctcagtcc ttttctgagg cataatggaa 1380
tctct gag t g 9=9 fcgagaccaaa 1440

gctcccagtc ttgtgacatt tgcaayy a . rtaaracca 1500

aaagtgaaag gaaagggggg aaaagggaga attctaaaaa tgcccatcct ctgaacacca 150
■ Stt 9 tgta ggcatctggg ggaggccagc tggggtgagg tcatctgcca .ccaggcc g

2 al tt g g Ucttgt ttatcacagc cacatgtggg gccactgcca gggcccgccc 1620

16

EP1 199 372 A2

caactctgca gtcattggag gagcttgaag
tcattttgca gttactggga gggggcttgc
gtccagctcc gcgcagggag ggaggctgtc
gttttccagt atgagacgaa caaagtcact
aagtggttct tccacgtgat catcttttcc
ccagatctct gcagtggccg acagcacaga
tctgaatctc acatggtttt cgaatctgag
gaggaagcag cagcaggcaa gaggaaacgg
acaagcggga ttcctttctg ctctacttca
cctggggaag gtaggaaagc gcagggcaac
ctcagggcac gcctggtgat catgctggca
ccaggcttga aaatgtgtta taactttagg
ttggtttttg tttcttctaa aagaaactta
acccatttaa agggtaccat ttaatgattt
cccacaatca attttagaat attttaatcg
tcatttccaa cactgttcct cctccttccc
tttgccgatt ctggacattt catataaatg
cttagcgtgt tttcaatgct catccatgtt
tttctttaaa gagacggggt ctcactattt
aagtgatctg cctgcctcgg cctcccaaag
ccggctgata cttcattcct ttttacggct
ttttttctat ccattcatcc attgatggac
tgaataatgc cacatgaaca tttgtgtaca-
tctcgaatat gtacctaaga gtaaaaattg
tggggggaat gtggagctga atttcacagc
agtatgaggg atccaatttc tccacatcct
ggttttgttg ttgtcatttt gtttttgtct
ctggagtgca gtggcgcaat cttggctcac
tctcctgcct cagcctcatg agtagctggg
atttttgtat ttttagtaga gatgaggttt
ctgacctcaa gtgatccgcc caccttggcc
cgccacgccc ggctgattgt ctgtcttttt
gtagttcatt gtggttgtga tttgaatttc
ctgattgata atgatgatga aggcaatttg
gagttagggt aacttatttc atagtactgg
tctgagcctc agtttctgca tctgttcata
catgaacagc ccttatcatg atgactgaca
ttttaaaaat aatcttttta agtctgggag
tgggaggccg aggcgggtgg atcacgaagt
gtgaaacccc atctctacta aaaatacaaa
aatcccagct actagggagg ctgaggcagg
cagtgagccg agatcaagcc actgcactcc
aaaataataa taatagtaat aatttttttg
acatgtatgt atttttatct atatcctctg
tcaggatttg aaatctggaa acgtggattc
tcatcatctg taaaatgggg agaattgttg
aagctgtttg agaaatatat ggcatatagt
taataatgct attattagga ttattattat

ttaaagactc ctgctaaaaa ccagtacgtt 1680
tgtggccctg tcaggaagag tagagctctg 1740
accatgccgg cctgctgcag ctgcagtgat 1800
cggatccaga gcatgaatta tggcaccatt 1860
tacgtttggt aagtgggatc tggggaggac 1920
aagccccagc gggcagcttc aggtgcacat 1980
acgtgctctc acagccagct gggcgggagg 2040
tgccaggctg cagcagagag aagccacagg 2100
ggcccgccag ggcgcgcaag gcagggcgtg 2160
accctggatc cccagggagg aggcgaggat 2220
tctgagtcac catgcttggg aggaatagga 2280
tcctcaccaa cgtcaggaag gccctgcttt 2340
ctgagatata atttatacac catacaattg 2400
tcagattatt cccagagttg tgcaaccatc 2460
actcaaaagg aatcccacac tccttcacca 2520
acccatcaat ttcctttctg cctctatgga 2580
gaatcacata atatgtggtc ctttgtggca 2640
gtagcatgtg ttgatacttc attcaatttt 2700
tgtccaggct ggtctcaaac tcctggactc 2760
tgtcaggatt acaggcgtga gccattgcac 2820
gagtagtact ccattgcatg gatagaccac 2880
attggggttg tttctctttt ttggctatca 2940
aggttttatg tggatatata ttctcctttc 3000
ctaggtcata tgttaactat gtttcacctt 3060
agctgcagtt ttttacattc ctatcagcag 3120
caccaacgct tgttatcgtc tgtctttttg 3180
ttgagatgaa gtcttgctct gttgcccagg 3240
tgcaacctcc acctccccgg ttcaagcgat 3300
attacaggtg tgcgtcacca ctcctcacta 3360
cgccatgtag gccaggctgg tctcaaactc 3420
tcccaaagtg ctgggattac aggcatgagc 3480
tattatagcc atgctagtgg gtgtgaagtg 3540
cctgatggtg agtgcctctt attctctgtg 3600
tatctataga gtggcagtgt agtttactaa 3660
ctatgtcttc tgggccaagt cattaacttc 3720
gggttgtggc aattaaccaa aaaaaaaagg 3780
taggataaga gctccataac tagtatctat 3840
tggtggctca cacctgtaat cccaacactt 3900
caggagtttg agaccagcct ggccaatatg 3960
aattagtggg gagtggtggt gcacacctgt 4020
agaatcgctt gaacccggag gcggaggttg 4080
agcctgggtg acagagcaag actccatctc 4140
attatataat agtatatatg tatataaaat 4200
ctctgaccct caaagtaacc acgtccaagt 4260
aaaaatcctt cacctctttg agccttggtt 4320
ataggaatat taaatgaact aataaatgca 4380
aatccctgat taagtgttag ttcttattat 4440
tcgattcata tgtttactgt tcaacaaata 4500

17

EP1 199 372 A2

ttgaatgata aacatatatg ctgggtccgg catggtggcc c— — - j£

„ ~ : = — — : tggt9ggtg .so

tgtg gtggaa actccatctg t g ctaaaaat ca gg a gg t gg 4740

cc tgt a atcc cagctactcg »~* « ^ ^ ctc 4800

aggttgcagt gagccaagat tgcaccactg a tatata tata gtatttttag 4860

tgtctcaaaa aa.aaaaaaa aaaatatata tatatatata tatata ^
tagagatggg gttttgccat ctcttatata tttttatatt

<210> 2

<211>

<212> DNA

<213> Homo sapiens

<400> 2 afl „ct atcaccatgc cggcctgctg cagctgcagt gatgttttcc 60

aaa acgcagg gaggg ag g ct gtcaccatg « tta t gg cacc attaagtggt 120

t cttccac g t gatcatcttt tccta= g t 9 ^ ^

agcggaaaga gcctgtcatc agttctgtgc » t gacaC cgc ag 300

aagaggafl at c^aat ggagtg^ a 9 ^

aaggCCaag a gcagcggttg tgtcccgag aggaat tca g a cc g ga agg t 480

— * taaaaag ": rXc ~ cccatc9aM 540

gtgtagtgca t g aa ggg aac ^ a ^acct g g g ttc actgtgctc a 600

cagt g g aa ga gg cccc=c gg cctgctctct tgaaca^ g ^

tcaaga acaa t a tc g acttc cccggccaca fl ' ttccga ctag 720

taaaca tc a c ttgtaccttc cacaagactc ^at ^cc ^ ^

gagaca tctt ccg a gaaac a ggcgat aa tt "tcagatg " tgccgtC cca 840

»— — ~ ^„ ,.o

~ at T o ;:: ::ir,r, —«,««,. •«■*— ' to

acttcagata cgccaagtac tacaagga artrtaaaat tt gacattatcc 1020

— — : :~ ™ ~: ~ 9 ^

a gct g g tt gt gtacatcggc tcaac ct ^
actt =ctcat egacact a tcca^ ^
agtg ctgtc a gccctgtgtg B te«a g tgaatccca c .ttaggatgg 1260

tggagccaaa gccgacatta aa gt a tgtgt cctttg gg » gtcccaagac 1320

tgaacc.gc. gctactaggg agaagtctgc ..g.tgtcaa ^ccaag «
c 9 tgcga tgga cttcacagat ttgtccaggc t = c_ a~cc^ ^
ttcctggac. a =c a gaggag at a c a gctgc «agaaagga gagagC c a ca 1500

a t a gccccgt ctggtgcc.g tgtggaagct gcctcccatc tcaactcc t g g g
ggt gcc t gga ggagctg.gc t g cc gga aa a agccggggg ^ * 1{20
tg ttc a ggaa gctggtcctg tccagacacg t cctgc a gtt «*~« gcctacaggC 1680
ccttgctggc gctggatgtg gattccacca ~»<^-~£^ ^L^ct 1740
„ c « „ a™ c— 1800

" r~ — — ttc 1853

18

EP1 199 372 A2

<210> 3
<211> 11266
<212> DMA

<213> Homo sapiens
<400> 3

catcacctac aaaggaaacc ccaaatccag tagcagtcac tccccattct ccccttcccc 60
tgtccctggc cacagtctac tttctgtctc tatagatgcc tattctggac atttcctata 120
aatagaattg tatatggtgt ggccttttgt gtctgtcttc tttcactcag catcatgttc 180
tccaggtcca tccatgttgt agcctgtgtc attgcttcat ccttcttatg gctaaataag 240
attctgtgta tgaatgtacc acattttatt tgtccattca tccgtcagtg gccacttgca 300
tggtttccac ttttttggcg attctgagta gtgctgctat aagcattcgt gtgcacattc 360
tggtggatat cgaatcactt ctccacatct tagtaacaca cgtcacttac tccccactct 420
gtcatccttc tatctgcagt atcccacccg caggacgctc tgttcctctg accgaggttg 480
taaaaaggga tggatggacc cgcagagcaa aggtaccttc tgtttctttt cccgagaccc 540
taggggtgga tggtctggca tcttggtgac atttgtgatg cccaggtcag gtcttcagcc 600
tctgctctca gctgccctct tccaccatca ccaagccata ggcgagtctg cccatgcttc 660
ggctctgtcc ccagcagacc agctgctgac tgtaaacatg actccagttt tccagtgaga 720
gaagaagctc ctaaaaacct agcaggttca ggattctaat cggtagaaaa ttcacatggc "780
ctatagcatc atctgagtat tctaaacttt ccccctgaat ttcctcaaag gttgaggacc 840
atgaactttt acccccaggg gaacctggca gcaataccca tattaacctg cagaattttt 900
tttgtttttt attttatttt attttttaaa cattttttgc actgttttat tttgattttg 960
attttgattt tatttatatc taagtgcagt gctattgcga tactgcagaa tttctttatc 1020
tcacatttta acttaaaaag gcacagggca gcgagcgcag aggctggtgc ctgtaatccc 1080
agcactttgg gagggtgagg cagatggatg cttgaggtca ggggttcgag aacagcctgg 1140
aaaacatggt gaaaccccgt ctctactaaa aatacaaaaa tcagccagac atggtggcac 1200
acgcttataa tcccagctac ttgggaggct gagacgtgag aatcacttga acctggaagg 1260
cagaggttgc agtgagccaa gatcatgcca ctgcactcca gcatgggtga cagagcgaga 1320
cccctttaaa aaaaaaaaaa aaaggcacag ggcaatttta aaaatactgc aaatagtaaa 1380
aaaaaaaaaa tcagtggtta taatgcaaac acacacaaaa aggcatatgc ccattactgc 1440
attctactcc atactgtatg tgtatttgag ttagtataaa agttatttta acattgctca 1500
ctatttaatt aattctccct tggaaactga ttaatcatcc tggcactcca ggaagatgtg 1560
ccatgctgat ttcatggctt tgcacatcct gggcaggctg tgtacccctt gagggacttg 1620
tgcccctttg agaggccatg ttctagtcca tttatactaa gtgagagcat acacctgttc 1680
cgctcccctc atgggcacct tttcttataa agaaacaaaa gagccagcag aatccacagt 1740
ctttctgtgt tctctctgat ctttattatg ttttgcttgt ttgccttgcc ttgtgttcgt 1800
tgtggttagg atgggcttga tggaagctga agctgcgtgg gttggaaagc ctggtcaaag 1860
cctagtctct cgcccgggtt gagttaatga tgtccctcct ggagaacgtc ctctctgcag 1920
ttctttcaca tctgtggttc tacgatgctt tgacccctat aggaattcag accggaaggt 1980
gtgtagtgca tgaagggaac cagaagacct gtgaagtctc tgcctggtgc cccatcgagg 2040
cagtggaaga ggccccccgg tgagtcgcat ggggagacag acacagtggc cctcagcggc 2100
gaccagatga ggccttgccg aggctgcttg ggccttcccc tctcagcaca gccctgcaaa 2160
gtcctgggtc ctaccggctt ggggacccct gcgctctgga tgcactgctt ggcacaaact 2220
agtatctctg ggagggccat ggtggttggt aaactgttgt aacactcctg taccaactgg 2280
taaatagcta ctaccctgag catccttggg tgtccctggc cccttccttc ccccagatct 2340
tccagggtac ccccagaccc cctcctgtag tgccacagca ggatcccttc tgacttgtca 2400
gtgtccatac tgagtgatca aggataggaa ggaaggaggg agatggaaag gaaggacgaa 2460
gcgaggaaag agaaggggaa ggggaggaaa aagcaaaagg ggtgagggta aaagaggggg 2520

19

EP 1 199 372 A2

. ttaaatactt acaatgacat acagatttgg tggtcccttg 2580

= = E:r.i = = = =

=== = E= = = = =

— =s = = =• -r- z
:= = := = = == z

agatagagtt ttgetatgtt geeeaggetg gttttgaaet ectgggetea agegatectc 3240
ctgeettgge cteeeaaagt gctggggtta eaggtttgag ccattgctcc cagcctgaat 3300

a tt taaa ttt aaatageeae aca t g t c t ag tggctaccat a« =
gcagttctag accgatgtga tteaggatca ttccctcagc atcgtggggc aaagagaaaa 3420
gcagttctag a g » gcgaaggttt cecaatgccg ggatgggggg 3480

ctgccccaag ctggcctgta gaaggctcag gcga as t . r ., mtLt ca.t 3540

tgcgctcagc agcatcaccc cttatgattc tcaatcgcta atagctccac tcaggttcat 3540
tgcg age * tttc tttgggaatc acccagctct gggagataca gcagcctcca 3600
ttcteggtea ggggcatttc tttggga* .^i-reaact 3660

ctcaggtagt ccttgttcaa gaeaagegge ccttgactga ctgcagtttc ^^ccagct 36
ctgctatcL ctcactcatt aaataaactg catctccagt gtgcctgcct ctgggct , a 3720

™ = = =: = = =

= : = = = = : === =
= = = = = = :

caggeggate acttgaggtc aggagtttga taccagcctg gcaaacatgg «aaa=ccca 414
tctctattaa aaaatgeata aattagccag gtgtggtggc acacgcctgt aatcctggct 4200
acCggagg ctgaggcagg agagtcgett gaacccggga gatggaggtt gcagtgacce
gagatcgege cactgcactc cagcctgggt gacagagtga gactccatct cagaaaaaaa

aaalaa aataaagaac tacctgagac caaacacttt acgaaaaaaa
ttgactcaca gotccacagg cttaacagga agectcagga gacttacaat -tggcagaa 444
ggcgaagggg aagcaaacac atcttaccat gatggagcag gaggegggtt tegggggatg
tgccgcacac CtC taaatga tcagatctcg tgagaactca ctcactatca cgagaacagc
alggaggaag tccgccccca tgattcagtc acctcccacc aggcccctcc tc ggcaca 2
gg a Lea attcaagatg agatttgggt ggg^caeag ageeaaaeca ^t~gat
caagaaggga gaaattette ttggaggagc tggagggget ttgtggagag tttcagaatg 4740
ctTgcccac taggtttget gtatccattt ctcttcatgc ateeeaaaga ccaagc a

ac agaag cctctggtce eaetggecca tgggctccct eggteeeeae eg.eaetaa
ggceattttg eatgtetete teeeaggeet getetettga aeagtgeega aaaetteaet 4920
rctcaU agatcaatat egae tt eeee ggccacaact acaccacgta ag t gcecagg
ctgcctggct gtettagtta tetaetgetg agtaataaat tateeeaaae cteagaagee 50
gaaadaca aacgectatt gtctcccacg gtttctgtgg gtcaggaatc tgggaatgac 10 0
tttgctgcgt ggttctggct caaggtctgt caggttgtag ccaagctgtc aaccagggct 5160
gcagtcattt ctaggcttga ctggggctgg agaacccttt teeaage.ee eaeacag g
ctcgtgggag ageteagtte eteaeeaegt gaaeetegee etagaeeaet tgagtatcct 28

"atltg tggctggctt ctcccagagc aagtgaccca agagagacag agcaagca
caagagtata aeeaagatgg aageeaeagt etttgggggg agaeeeeaae aettetgeea

20

EP 1 199 372 A2

tatgccattg gtcacacaga tcaaccctgg tccagtgtga gaggccactg cccaggggtc 5460
ccaggaggca gggatcattt ggggctttca tggaacctct ccaccacact ggctcactcc 5520
tgggaaagag acagatctgt tttcaatcga gatgtttgtt tgtttgcttt taattatgga 5580
caggagaaac atcctgccag gtttaaacat cacttgtacc ttccacaaga ctcagaatcc 5640
acagtgtccc attttccgac taggagacat cttccgagaa acaggcgata atttttcaga 5700
tgtggcaatt caggttggtg gtgctttgta cactgggatg tggggctgtg tgtctaggga 5760
tggaggatgt caaacagcca agaggccggg ccactgggtc ttcataatgt ggctcacatt 5820
tactgagcat ttagtaaatc cacccgctgc gctaaggtct ttacctacgc tacctcgtca 5880
aatcccaaaa caatccttat gagtgagagc tacttggtgt attcctttcc tgtggctgct 5940
gtagcaagtt atcaaagctt agtggcttca aacaacacat atttgcttat gttgccagag 6000
atcagaacjtt ggagatgatt ttccctgagc cagggcggtg ctccctctgg gactttaagg 6060
gagaatccag ttcctcagct tttccacctt ctggagctgc attccttgca tttcttcaaa 6120
gccagcagca taacatcttg cctcagtggc cactttcact ccctatcctg tgtccaatct 6180
ccctttgcct ctgtcttaaa aagagagaga gcatttacaa gagggggcat ttaaggacca 6240
actggataat ccaggataat ctcccatctc aagatccttc atttaggctg ggcacggtgg 6300
ctcatgcctg taatcccagc actttgggag gctgaggtgg gtggatcacc tgaggtcagg 6360
agttcaacac cagcctggcc aacatggtga aagcccatct ttactaaaaa tacaaaaaaa 6420
aaaaaaaaat agccgggcat gattgcgggc tcctgtaatc ccagctactc gggaggctga 6480
gacaggagaa tcgcttgaac ctgggaggca gaggttgcag tgagccgaga tcgcaccact 6540
gcactccagc ctaggtgaca agagcgaaac tccatctcaa aaaaaaaaaa aaaatccttc 6600
atgtattcgc atctgcaaag agctttccct aggggagtac taggaggtaa agcagaaaag 6660
atatttgata gagtgccctg aattccagtc taataagttt ggacttgatc tttaatgggg 6720
gcgtgggggg cattaaaggt gtttgggtac aggagtggtc tgttgaaagt tgtattttag 6780
gacaatgagt ttaacagtga tgtgtcccag acgggggtag ggagagtgag gagatgcgat 6840
tgtggctgcc acaataacac ttgtgcgagt taggtggggc tgtacatatg gttcttcaat 6900
cagcattttt tctctaaaaa ccttaagcaa tcctggctat gcagggagat gtctggcggt 6960
tgcgtaactc acacccagca gccatagaga ctgtcccttg ttgatccttc agggcggaat 7020
aatgggcatt gagatctact gggactgcaa cctagaccgt tggttccatc actgccgtcc 7080
caaatacagt ttccgtcgcc ttgacgacaa gaccaccaac gtgtccttgt accctggcta 7140
caacttcagg taactccaag gcccaggtca aactcaccca gtggctgaat cgcattccca 7200
ggaactggtg agactaattt tggtttccaa ggcaacaaga tgaatgaaaa aagactttct 7260
ctaagaacta ggtgataact gaattttttc cataattttt taaaattctc aaaagagata 7320
cactctttat tttttactta tttttttttt tttgaaatgg agtctcactc tgtcacccag 7380
gctgaagtgc agtggcgcca tctcagtcac tgcaaacttc cgcctcccag gttcaagcga 7440
ctctcctgcc tcagcctccc aagtagctga gattacaggc ggatgcacac tgtttataaa 7500
acaaaactat tgggaaacag aaaagcatag agggggatca aaatcgccca taattcccct 7560
accctgaaat aatcaataac aaccctcggg ggaattttcc tcatctgtac caattatttc 7620
atacagctcc tatgagatca tagcatatat atatatatat cttgtggtat tctgcagggt 7680
ttttcatacc acagccactc aaaattcttt gtaaccatca cattaatgat cataacattc 7740
cattttgtag gtgaacaaat aacaactgct acaattcagg aagtgttttc ttttcttttc 7800
ttttcttttc tttttttttt ttagatggag tcacactctg cttgcccagg ctggagtgca 7860
gtggcatgat ctcagctcac tgcaacctct gcctcctagg tccaagcgat cctcccacct 7920
cccaagtttc tgggaccaca ggcatgtgcc accacaccca gctaattttt gtatattcag 7980
tagagatggg gtttcactgt gttggccagt ctggtctcga actcttgacc tcaagtgatc 8040
ttcccacctt ggcttcccaa agtgctagga ttacagtcat gagccactgt gcctggccca 8100
aggagggttt tccatatacc aagcactccc catcgccatc cctaaatctc ccaacaaccc 8160
tggaaggaag atattgtttc tggaagatga tttgcccaag acccacagct gatagtacat 8220
gtttgcataa ttctaaccca cgttcactct gaccccacac tcacactccc atcccttccc 8280

21

EP1 199 372 A2

► rtttc tcaccgtacg cctccatgaa ttgaatattt gagttgcttc 8340
ttcccatctc aatgattttc ^accgtacg caccgtaaac ttcttctttg 8400

ccagtttttc tagtacaagt aaccacag g tgcatctt g MH

— ~: =. — « 652 o

cattttatca tatgcttttt attt« _ fl g^cgggtgc aattatagct 8580

ttct gagaca gagtctcact ctgccaccca «^^. ctgagtagct 3640

cact gcaacc tctgcctccc aggcccaagt J gg 8700

ag gac t a=ag gtgcatgcca ccatgcccag «tt£ , ^ ^

ggc ctcctaa agcgttggga ttgcagg^ g ^

ttctaggatt tttatttggt gctttttcaa ■ „ tat cattcc tttcataccg 8940

tt ctta=ct t aaggatccta ctccttctgt ccattctact ^atcattcc

acctattatc tgaagtaact tgggtgggag ttctcctcgt gggctttgaa atactg

— — = = rs ~ t 9120
— ;~ — — : — =

I tt atgcc t3 taatcccagc actttgggag gccaagg^ gag^act «

— — C " "a .-gaggca 9360

aacatcagcc aggtgtgggg gcatgcacct ctgg c g gC cactgcac 9420

— «- — - ™: — : -

tccagcctgg gtgacagagt gagaccctta ca gg gg gcC gctcctt 9S40

agt gg t cc t a tttagaaagg ggctggactt c^«~ - - g g ^

a g t gcttata cctggcccac atcactcatt "*gt«tct gc gg 9660

ttt gagt tt g aaacccttga ctcaaaggca ggctgatgct * « ^

t993a T t " = ~ — ~c 9,80

i™ := g~ = — ::::

taccaatcag gtgtcagcaa gtgtccttcc agcgactgag tgttctttt a 9960

t=c t ggagag acctgagccc tct.aaggcc tcagccagtt atgat^taa g ^

= = = = =: ::= s

=======

= = = = = s=

aaca ac t taa ata.at.aat a aa ttgt tga ggtctgatga gtaagtgg g
cca g= aga =a =ac aa aagag aaggaaa^a ca*g^ <J ^

actttct aaa acataggaag = - ^ ^ atgcacgacc 10560
aaatgacctg tggcgaaatg tccttattca gcg gg cagcaggagt 10620

t g t g t gaag t ggatcaggcc acccagaatg cacga gcgc ctcaggcc ag J ^
atgtgtctgt gttaatttcc tgtggctatt atgactaatt gccacaaatg tgg gg
iraac/ga aa tt aa tCtt c tt a C ag tt c tggaagccag a = ~ «

==== = = = ==5=

= = = = = = = = = =

= = = = = = -

ttaattatat ctgcaaacac tgtaccccca *<»v y *

22

EP1 199 372 A2

agatacttga acatatctta tttgggggct caacccattc cagtgtacga aaaacactct 11220
tgttcaaggc ccgatgtttc tcagggcata gcccactgac tacctg 11266

<210> 4

<211> 595

<212> PRT

<213> Homo sapiens

<400> 4

Met Pro Ala Cys Cys Ser Cys Ser Asp Val Phe Gin Tyr Glu Thr Asn
15 10 15

Lys Val Thr Arg lie Gin Ser Met Asn Tyr Gly Thr lie Lys Trp Phe
20 25 30

Phe His Val He He Phe Ser Tyr Val Cys Phe Ala Leu Val Ser Asp
35 40 45

Lys Leu Tyr Gin Arg Lys Glu Pro Val He Ser Ser Val His Thr Lys
50 55 60

Val Lys Gly He Ala Glu Val Lys-Glu Glu He Val Glu Asn Gly Val
65 70 75 80

Lys Lys Leu Val His Ser Val Phe Asp Thr Ala Asp Tyr Thr Phe Pro
85 90 95

Leu Gin Gly Asn Ser Phe Phe Val Met Thr Asn Phe Leu Lys Thr Glu
100 105 110

Gly Gin Glu Gin Arg Leu Cys Pro Glu Tyr Pro Thr Arg Arg Thr Leu
115 120 125

Cys Ser Ser Asp Arg Gly Cys Lys Lys Gly Trp Met Asp Pro Gin Ser
130 135 140

Lys Gly He Gin Thr Gly Arg Cys Val Val His Glu Gly Asn Gin Lys
145 150 155 160

Thr Cys Glu Val Ser Ala Trp Cys Pro He Glu Ala Val Glu Glu Ala
165 170 175

Pro Arg Pro Ala Leu Leu Asn Ser Ala Glu Asn Phe Thr Val Leu He
180 185 190

Lys Asn Asn He Asp Phe Pro Gly His Asn Tyr Thr Thr Arg Asn He
195 200 205

23

Leu Pro Gly Leu Asn
210

EP 1 199 372 A2

He Thr Cys Thr Phe His Lys Ar Gin Asn Pro

215

220

Cl „ Cys Pro lie Phe Arg Leu GXy Asp He Phe Arg Glu Thr Oly Asp
225 230 " 5

Asn Phe Ser Asp VaX Ala lie Gin Gly Gly He Met Gly He Glu He
245 250 2 55

~ t»u asp Ar<J Trp Phe His His Cys Arg Pro Lys

Tyr Trp Asp Cys Asn Leu Asp Arg

260 265 270

Tyr Ser Phe Arg Arg Leu Asp Asp Lys Thr Thr Asn Val Ser Leu Tyr
275 2 80 285

Pro Gly Tyr Asn Phe Arg Tyr Ala Lys Tyr Tyr Lys Glu Asn Asn Val
oqc. 300
290 29b

Olu Lys Arg Thr Leu He Lys Val Phe Gly He Arg Phe Asp He Leu
305 310 315

Val Phe Gly Thr Gly Gly Lys Phe- Asp He He Gin Leu Val Val Tyr
325 "0 "5

He Gly Ser Thr Leu Ser Tyr Phe Gly Leu Ala Ala Val Phe He Asp
340 345 350

Phe Leu lie Asp Thr Tyr Ser Ser Asn cys Cys Arg Ser His He Tyr
355 360 3 "

Pro Trp cys Lys cys Cys Gin Pro Cys Val Val Asn Glu Tyr Tyr Tyr

370 3 "
Arg Lys Lys Cys Glu Ser He Val Glu Pro Lys Pro Thr Leu Lys Tyr
385 390 395

r., m« He Ara Met Val Asn Gin Gin Leu

Val Ser Phe Val Asp Glu Ser His lie Arg net

405 415

Leu Gly Arg Ser Leu Gin Asp Val Lys Gly Gin Glu Val Pro Arg Pro
420 430

Ala Met Asp Phe Thr Asp Leu Ser Arg Leu Pro Leu Ala Leu His Asp

435

440

445

Thr Pr o Pro He Pro Gly Gin Pro Glu Glu He Gin Leu Leu Arg Lys
455 460

450

24

EP1 199 372 A2

Glu Ala Thr Pro Arg Ser Arg Asp Ser Pro Val Trp Cys Gin Cys Gly
465 470 475 480

Ser Cys Leu Pro Ser Gin Leu Pro Glu Ser His Arg Cys Leu Glu Glu
485 490 495

Leu Cys Cys Arg Lys Lys Pro Gly Ala Cys lie Thr Thr Ser Glu Leu
500 505 510

Phe Arg Lys Leu Val Leu Ser Arg His Val Leu Gin Phe Leu Leu Leu
515 520 525

Tyr Gin Glu Pro Leu Leu Ala Leu Asp Val Asp Ser Thr Asn Ser Arg
530 535 540

Leu Arg His Cys Ala Tyr Arg Cys Tyr Ala Thr Trp Arg Phe Gly Ser
545 550 555 560

Gin Asp Met Ala Asp Phe Ala lie Leu Pro Ser Cys Cys Arg Trp Arg
565 570 575

lie Arg Lys Glu Phe Pro Lys Ser- Glu Gly Gin Tyr Ser Gly Phe Lys
580 ' ' 585 590

Ser Pro Tyr
595

Claims

1. A method for the diagnosis of a polymorphism in P2X 7 in a human, which method comprises determining the
sequence of the human at one or more of the following positions:

positions 936, 1012, 1147, 1343 and 1476 in the 5'UTR region of the P2X 7 gene as defined by the position in
SEQIDNO:1;

positions 253, 488, 489, 760, 835, 853, 1068, 1096, 1315, 1324, 1405, 1448, 1494, 1513, 1628 and 1772 in
the coding region of the P2X 7 gene as defined by the position in SEQ ID NO: 2; and
positions 4780, 4845, 4849, 5021 , 5554, 5579, 5535, 5845 and 6911 in the intron region of the P2X 7 gene as
defined by the position in SEQ ID NO: 3;

positions 76 ,155, 245, 270, 276, 348, 357, 430, 433, 460, 490 and 496 in the P2X 7 polypeptide as defined
by the position in SEQ ID NO: 4;

and determining the status of the human by reference to polymorphism in P2X 7 .

2. Use of a diagnostic method as defined in claim 1 to assess the pharmacogenetics of a drug acting at P2X 7 .

3. A polynucleotide comprising at least 20 bases of the human P2X 7 gene and comprising an allelic variant selected
from any one of the following:

25

EP 1 199 372 A2

Region 1

/ariantSEQ IDNO:1 I

5'UTR

936 A

1012C

1147 G

1343 A

1476 G

Region

Variant <>FQ ID NO'. 2

exon 2

253 C

exon 5

488 A

489 T

exon 7

760 G

exon 8

835 A

853 A

exon 11

1068 A

1096 G

exon 12

1315G

exon 13

1324T

1405G

1448T

1494G

1513C

1628T

1772 A

Region

Variant SEQ ID NO: 3

intron E

4780 T

4845 T

4849 C

intron F

5021 C

5554 (GTTT) n ,n=4

5579 C

5535 T

intron G

5845 T

6911 C

A nucleotide primer which can detect a polymorphism as defined in claim 1 .
An allele specific primer capable of detecting a P2X 7 gene polymorphism as defined in claim 1 .
An a.,e.e-specific oligonucleotide probe capable of detecting a P2X 7 gene polymorphism as defined in claim 1 .
Use of a P2X 7 gene polymorphism as defined in claim 1 as a genetic marker in a linkage study.
A method of treating a human in need of treatment with a drug acting at P2X 7 in which the method comprises:
0 diagnosis of a polymorphism in P2X 7 in the human, which diagnosis preferably comprises determining the

26

EP1 199 372 A2

sequence at one or more of the following positions:

positions 936, 1012,11 47, 1 343 and 1 476 in the 5'UTR region of the P2X 7 gene as defined by the position
in SEQ ID NO: 1;

5 positions 253, 488, 489, 760, 835, 853, 1068, 1096, 1315, 1324, 1405, 1448, 1494, 1513, 1628 and 1772

in the coding region of the P2X 7 gene as defined by the position in SEQ ID NO: 2; and
positions 4780, 4845, 4849, 5021 , 5554, 5579, 5535, 5845 and 691 1 in the intron region of the P2X 7 gene
as defined by the position in SEQ ID NO: 3; and

positions 76 ,1 55, 245, 270, 276, 348, 357, 430, 433, 460, 490 and 496 in the P2X 7 polypeptide as defined
10 by the position in SEQ ID NO: 4;

and determining the status of the human by reference to polymorphism in P2X 7 ; and

ii) administering an effective amount of the drug.

15 9. An allelic variant of human P2X 7 polypeptide comprising at least one of the following:

a alanine at position 76 of SEQ ID NO 4;

a tyrosine at position 155 of SEQ ID NO 4;

a glycine at position 245 of SEQ ID NO 4;
20 a histidine at position 270 of SEQ ID NO 4;

a histidine at position 276 of SEQ ID NO 4;

a threonine at position 348 of SEQ ID NO 4;

a serine at position 357 of SEQ ID NO 4;

a arginine at position 430 of SEQ ID NO 4;
25 a valine at position 433 of SEQ ID NO 4;

a arginine at position 460 of SEQ ID NO 4;

a glycine at position 490 of SEQ ID NO 4; and

a glutamic acid at position 496 of SEQ ID NO 4;

30 or a fragment thereof comprising at least 1 0 amino acids provided that the fragment comprises at least one allelic

variant.

10. An antibody specific for an allelic variant of human P2X 7 polypeptide as defined in claim 9.
35 11. A polynucleotide comprising any one of the following twenty six P2X 7 haplotypes:

40

45

50

55

1012

489

5579

835

853

1068

1096

1405

1513

SEQ ID
1

SEQ ID

2

SEQ ID

3

SEQ ID

2

SEQ ID 2

SEQ ID 2

SEQ ID 2

SEQ ID 2

SEQ ID 2

1

T

T

C

G

G

A

G

A

A

2

C

C

G

G

G

G

C

A

A

3

C

C

C

A

G

G

C

A

C I

4

c

T

G

G

G

A

C

G

A

5

c

C

G

G

G

A

G

A

A

6

c

C

C

A

G

G

C

A

A

7

T

T

G

G

G

A

C

G

A

8

c

T

C

G

G

G

C

A

A

9

c

C

C

G

G

A

C

A

A

10

c

T

G

G

G

G

C

A

C

11

T

C

G

G

G

A

c

A

A

! 12

c

T

C

G

G

G

c

A

C

27

EP1 199 372 A2

(continued)

12. A human P2X 7 polypeptide comprising one ,rf ihr following
amino acids based on positions identified in SEQ ID NO. 4.

30

35

40

45

eighteen combinations of alleleic variant determined

50

55

155

270

276

348

357

460

496

1 1

Y

R

R

T

S

Q

E

I 2

YR

R

T

T

R

E

I 3

Y

R

R

T

T

Q

E

4

Y

R

R

T

S

R

E

J 5

Y

R

R

A

T

Q

A

I 6

Y

R

R

A

T

Q

E

J 7

Y

R

R

A

S

Q

E

I 8

Y

R

H

T

s

R

E

I 9

Y

H

R

A

T

Q

E

I 10

H

R

R

T

T

Q

E

11

H

R

R

T

T

R

E

J t2

H

R

R

A

T

Q

A

t3

H

R

R

A

S

Q

E

14

H

R

R

A

T

Q

E

15

H

R

R

T

S

Q

E

16

H

H

R

A

T

Q

A

17

H

H

R

A

T

Q

E

18

I H I H

I H

I A

T | Q

E

28

EP1 199 372 A2

13. A polynucleotide which encodes any human P2X 7 polypeptide as defined in claim 12.

5
10
15
20
25
30
35
40
45
50

29

(19)

(12)

JEuropai!
Europea
Office e

Europaisches Patentamt
European Patent Office

uropeen des brevets

(1D EP 1 199 372 A3

EUROPEAN PATENT APPLICATION

(88) Date of publication A3:

12.05.2004 Bulletin 2004/20

(43) Date of publication A2:

24.04.2002 Bulletin 2002/17

(21) Application number: 01308837.2

(22) Date of filing: 17.10.2001

(51) lntCl7: C12Q 1/68, C07K 16/28,
C07K 14/705, C12N 15/12

(84) Designated Contracting States:

AT BE CH CY DE DK ES Fl FR GB GR IE IT LI LU
MC NL PT SE TR
Designated Extension States:
AL LT LV MK RO SI

(30) Priority: 21.10.2000 GB 0025859
06.04.2001 GB 0108654
02.11.2000 US 244897 P

(72) Inventor: Morten, John Edward Norris
Cheshire SK10 4TG (GB)

(74) Representative: Giles, Allen Frank et al
AstraZeneca,

Global Intellectual Property Patents,
Mereside,
Alderley Park

Macclesfield, Cheshire SK10 4TG (GB)

(71) Applicant: AstraZeneca AB
151 85 Sddertalje (SE)

(54) Polymorphisms in the human P2X7 gene

(57) This invention relates to polymorphisms in the
human P2X 7 gene and corresponding novel allelic
polypeptides encoded thereby. The invention also re-
lates to methods and materials for analysing allelic var-

iation in the P2X 7 gene, and to the use of P2X 7 polymor-
phism in treatment of diseases with P2X 7 drugs.

CO
<
CM

CO

o>

CL

LU

Printed by Jouve. 75001 PARIS (FR)

EP1 199 372 A3

European Patent
Office

PARTIAL EUROPEAN SEARCH REPORT

Application Number

which under Rule 45 of the European Patent Convention^ p 01 30 8837
shall be considered, for the purposes of subsequent
proceedings, as the European search report

DOCUMENTS CONSIDERED TO BE RELEVANT

Category

Citation of document with indication, where appropriate,
ot relevant passages

Relevant
to claim

CLASSIFICATION OF THE
APPLICATION (lnLCI.7)

X

WO 97 40462 A (SPECTRA BIOMEDICAL INC)
30 October 1997 (1997-10-30)
* abstract; claim 1 *

1-7

C12Q1/68C07K16/2
8

C12Q1/68

x

HALUSHKA M K ET AL: "Patterns of
single-nudeotide polymorphisms in
candidate genes for blood-pressure
homeostasis."

NATURE GENETICS. UNITED STATES JUL 1999,
vol. 22, no. 3, July 1999 (1999-07), pages
239-247, XP000985696
ISSN: 1061-4036
* page 239 *

1-7

X

BROOKES A J: "The essence of SNPs."

GENE. NETHERLANDS 8 JUL 1999,

vol. 234, no. 2, 8 July 1999 (1999-07-08),

pages 177-186, XP004173090

ISSN: 0378-1119

* the whole document *

1-7

-/-

TECHNICAL FIELDS
SEARCHED flnt.CI.7)

C12Q

INCOMPLETE SEARCH

The Search Division considers thai the present application, or one or more of its claims, does/do
not comply with (he EPC to such an extent that a meaningful search into the stale or the an cannot
be carried out. or can only be carried out partially, for these claims.

Claims se

arched compfeteiy :

Claims se

arched incompletely :

Claims not searched :

Reason for the limitation of the search:

see

sheet C

Ptac© of search

MUNICH

Date of compter fcjn of the search

12 March 2004

Costa Roldan, N

CATEGORY OF CfTEO COCUMENTS

X : part iculany relevant if taxen atone

Y : part icularly relevant if combined wltti another

document of the same category
A : technological background
O : non-written disclosure
P : intermediate document

T : theory or principle underlying the invention
E : earlier patent document, but published on. or

after the fifing dale
D : document cited in the application
L : document cried tor other reasons

& : member of the same patent tamfly . corresponding

2

EP1 199 372 A3

J Application Number
European Patent INCOMPLETE SEARCH Ef> Q1 3Q mJ

Office SHEET C

Claim(s) searched completely:
1-7,9-13

Claim(s) searched Incompletely.

Reason for the limitation of the search (non-patentable invention(s)):
Article 52 (4) EPC - Method for treatment of the human or animal body by
therapy

Further limitation of the search

Claim(s) searched completely:
1-3,5-7,9-13

Claim(s) searched incompletely:
4

Claim(s) not searched:
8

Reason for the limitation of the search:

Calm 4 is directed to a prime; - which primer
-^-51 »!c 1 ^Wjirlraf^ been
seXed^

in length which are complementary or identical io v
gene.

Claim 8 «. ... 'LlV^ncS.S "2f ^ SSw

that no search is possible.

EP1 199 372 A3

European Patent
Office

PARTIAL EUROPEAN SEARCH REPORT

Application Number

EP 01 30 8837

DOCUMENTS CONSIDERED TO BE RELEVANT

Category

X
A

D,A

2

Citation of document with indication, where appropriate,
of relevant passages

US 6 133 434 A (BUELL GARY NUTTER ET AL)
17 October 2000 (2000-10-17)

* column 35 - column 38; claim 7 *

* column 37 - column 40; claim 1 *

BUELL 6 N ET AL: "Gene structure and
chromosomal localization of the human P2X7
receptor . "

RECEPTORS & CHANNELS. SWITZERLAND 1998,
vol. 5, no. 6, 1998, pages 347-354,
XP009021403
ISSN: 1060-6823

* the whole document *

W0 99 29660 A (ASTRA PHARMA PROD
;CLADINGBOEL DAVID (GB); M0RTIM0RE MICHAEL
(GB);) 17 June 1999 (1999-06-17)

* abstract *

LYNCH K J ET AL: "MOLECULAR AND
FUNCTIONAL CHARACTERIZATION OF HUMAN P2X2
RECEPTORS"

MOLECULAR PHARMACOLOGY, BALTIMORE, MD, US,
vol. 56, no. 6, December 1999 (1999-12),
pages 1171-1181, XP000876836
ISSN: 0026-895X

* page 1171 *

Relevant
tociaim

4,9

1-3,5-7,
10-13

1-3,5-7,
9-13

1-7,9-13

1-7,9-13

CLASSIFICATION OF THE
APPLICATION (lnLCL7)

TECHNICAL FIELDS
SEARCHED (intci.7)

4

EP 1 199 372 A3

European Patent
Office

PARTIAL EUROPEAN SEARCH REPORT

Application Number

EP 01 30 8837

nggUMENTS CONSIDERE D TO BE RELEVANT

Category

P.X

Relevant
to claim

Citation ot document with Indication , where appropriate,
of relevant passage s . — -

WILEY J S ET AL: "GENETIC POLYMORPHISMS |l-7,9-13
OF THE HUMAN P2X7 RECEPTOR AND
SS T gSSA F SSBE. NEW YORK, NY,

Sol 63, no. 2/3, June 2001 (2001-06),
pages 72-76, XPO01119468
ISSN: 0272-4391
* page 72; table 1 *

Irn REN J ET AL- "A Glu-496 to Ala |l-7,9-13

SlKrJhl- 1«* to loss of function of

the human P2X7 receptor
I JOURNAL OF BIOLOGICAL CHEMISTRY,

vol. 276, no. 14,

6 April 2001 (2001-04-06), pages

11135-11142, XP002263729

ISSN: 0021-9258

* the whole document *

CLASSIFICATION OF THE
APPLICATION (lm.Ct.7)

TECHNICAL FIELDS
SEARCHED (InLCLT)

5

EP1 199 372 A3

European Patent
Office

EP 01 30 8837

Application Number

CLAIMS INCURRING FEES

The present European patent application comprised at the time of filing more than ten claims.

□ Only part of the claims have been paid within the prescribed time limit. The present European search
report has been drawn up for the first ten claims and for those claims for which claims fees have
been paid, namely claim(s):

□ No claims fees have been paid within the prescribed time limit The present European search report has
been drawn up for the first ten claims.

LACK OF UNITY OF INVENTION

The Search Division considers that the present European patent application does not comply with the
requirements of unity of invention and relates to several inventions or groups of inventions, namely:

see sheet B

□ All further search fees have been paid within the fixed time limit The present European search report ha*
been drawn up for all claims.

□ As all searchable daims could be searched without effort justifying an additional fee, the Search Division
did not invite payment of any additional fee.

Only part of the further search fees have been paid within the fixed time limit. The present European
search report has been drawn up for those parts of the European patent appBcation which relate to the
inventions in respect of which search fees have been paid, namely daims:

□ None of the further search fees have been paid within the fixed time limit The present European search
report has been drawn up for those parts of the European patent application which relate to the invention
first mentioned in the daims. namely claims:

1-13 (all partially), inventions 1 and 28

6

EP 1 199 372 A3

European Patent LACK OF UNITY OF INVENTION
Office SHEET B

Application Number

EP 01 30 8837

Inventions 1: claims 1-8 (partially)
Invention 1

a S m e?hod for the diagnosis of said polymorphism for
determining the status of a human.

Invention 2-16: claims 1-8 (partially)
Inventions 2 to 16

6911.

Inventions 17-2V. claims 1-10 (partially)
Invention 17

c 1 1 n " ?«; 9 e stud,, probes

a human.

Inventions 18 to 21

ibid for SNPs at nucleotide positions:

760 (the allelic variant of h«»n P2X7 polypeptide
comprising a Glycine at position 245 of SEQ ID N0.4),

1315 (the allelic variant of human P2X7 P°lyP e Pt 1 ^.
comprising an Arglnine at position 430 of SEQ ID 10:4).

1324 (the allelic variant of human P2X7 P°lyP e Pt1<je
comprising a Valine at position 433 of SEQ 10 N0:4),

1/104 ftho allelic variant of human P2X7 polypeptide
JSrisKo « Glycine at position 490 of SEQ ID N0:4)

EP1 199 372 A3

European Patent
Office

LACK OF UNITY OF INVENTION
SHEET B

EP 01 30 8837

Application Number

The Search Division considers that the present European patent application does not comply with the
requirements of unity of invention and relates to several inventions or groups of inventions, namely:

Inventions 22-28: claims 1-13 (partially)
Invention 22

A polynucleotide comprising at least 20 bases of the human
P2X7 gene comprising the allelic variant 489 T; an allelic
variant of human P2X7 polypeptide comprising an Tyrosine at
position 155 of SEQ ID N0:4; the polynucleotide which
encodes said polypeptides; the use of said polymorphism as a
genetic marker in a linkage study, probes and primers for
the detection of said polymorphism; and a method for the
diagnosis of said polymorphism for determining the status of
a human.

Invention 23 to 28

ibid for SNPs at nucleotide positions:

835 (the allelic variant comprising a Histidine at position
270 of SEQ ID NO: 4),

853 (the allelic variant comprising a Histidine at position
276 of SEQ ID N0:4)

1068 (the allelic variant comprising a Threonine at position
348 of SEQ ID NO: 4)

1096 (the allelic variant comprising a Serine at position
357 of SEQ ID NO: 4)

1405 (the allelic variant comprising Arginine at position
460 of SEQ ID NO: 4)

1513 (the allelic variant comprising a Glutamic acid at
position 496 of SEQ ID N0:4)

Inventions 29-30 : claims 1-8 and 11 (partially)
Invention 29

A polynucleotide comprising at least 20 bases of the human
P2X7 gene comprising the allelic variant 1012 C; the use of
said polymorphism as a genetic marker In a linkage study,
probes and primers for the detection of said polymorphism;
and a method for the diagnosis of said polymorphism for
determining the status of a human.

8

EP 1 199 372 A3

European Patent LACK OF UNITY OF INVENTION
Office SHEET B

Application Number

EP 01 30 8837

Invention 30

A polynucleotide comprising at least : a > bases of the jj*
P2X7 gene comprising the allelic variant 5579^,
said polymorphs as a genetic mar*e po^orphism;

determining the status of a human.

>

EP 1 199 372 A3

ANNEX TO THE EUROPEAN SEARCH REPORT
ON EUROPEAN PATENT APPLICATION NO.

EP 01 30 8837

This annex fists the patent family membersrelating to the patent documents cited In the above-mentioned European search report
The members are as contained in the European Patent Office EDP lile on

The European Patent Office Is in no way liable for these particulars which are merely given for the purpose of in formation.

12-03-2004

Patent document

Publication

Patent family

Publication

cited in search report

date

member(s)

date

A
M

AU

2734197 A

EP

0897567 A2

JP

2000508912 T

1 o m onnn
I0-U/-ZUUU

WO

ft "7 A ft AC O

9/4U46Z

AZ

0 r\ 1 r\ 1 next

30-10-1997 j

US 6133434

A

17-10-2000

US

6509163

Bl

21-01-2003

W0 9929660

A

17-06-1999

AT

234274

T

15-03-2003

AU

746716

B2

rtO AC 1AAO

02-05-2002

AU

1791499

A

28-06-1999

BR

9813368

A

03-10-2000

CA

2312889

Al

17-06-1999

CN

1280560

T

17-01-2001

DE

69812159

01

17-04-2003

DE

69812159

T2

18-12-2003

DK

1036058

T3

30-06-2003

EE

200000320

A

15-08-2001

EP

1036058

Al

20-09-2000

ES^

2195433

T3 .

01-12-2003

HK

1028594

Al

05-09-2003

HU

0100431

A2

30-07-2001

JP

2001525391

T

11-12-2001

NO

20002785

A

01-08-2000

NZ

504375

A

29-08-2003

PL

340890

Al

12-03-2001

PT

1036058

T

31-07-2003

RU

2197477

C2

27-01-2003

WO

9929660

Al

17-06-1999

SK

8412000

A3

07-11-2000

TR

200001558

T2

23-10-2000

US

6242470

Bl

05-06-2001

i For more detafls about this annex : see Official Journal of the European Patent Office, No. 12/82

10

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